Stimulation of protein kinase C-dependent and -independent signaling pathways by bistratene A in intestinal epithelial cells

The marine toxin bistratene A (BisA) potently induces cytostasis and differentiation in a variety of systems. Evidence that BisA is a selective activator of protein kinase C (PKC) delta implicates PKC delta signaling in the negative growth-regulatory effects of this agent. The current study further investigates the signaling pathways activated by BisA by comparing its effects with those of the PKC agonist phorbol 12-myristate 13-acetate (PMA) in the IEC-18 intestinal crypt cell line. Both BisA and PMA induced cell cycle arrest in these cells, albeit with different kinetics. While BisA produced sustained cell cycle arrest in G(o)/G(1) and G(2)/M, the effects of PMA were transient and involved mainly a G(o)/G(1), blockade. BisA also produced apoptosis in a proportion of the population, an effect not seen with PMA. Both agents induced membrane translocation/activation of PKC, with BisA translocating only PKC delta and PMA translocating PKC alpha, delta, and epsilon in these cells. Notably, while depletion of PKC alpha, delta, and epsilon abrogated the cell cycle-specific effects of PMA in IEC-18 cells, the absence of these PKC isozymes failed to inhibit BisA-induced G(o)/G(1), and G(2)/M arrest or apoptosis. The cell cycle inhibitory and apoptotic effects of BisA, therefore, appear to be PKC-independent in IEG-18 cells. On the other hand, BisA and PMA both promoted PKC-dependent activation of Erk 1 and 2 in this system. Thus, intestinal epithelial cells respond to BisA through activation of at least two signaling pathways: a PKC delta -dependent pathway, which leads to activation of mitogen-activated protein kinase and possibly cytostasis in the appropriate context, and a PKC-independent pathway, which induces both cell cycle arrest in G(o)/G(1) and G(2)/M and apoptosis through as yet unknown mechanisms. (C) 2001 Elsevier Science Inc. All rights reserved.

Simultaneous administration of a cocktail of markers to measure renal drug elimination pathways: absence of a pharmacokinetic interaction between fluconazole and sinistrin, p-aminohippuric acid and pindolol

Aims Previous studies suggest that estimated creatinine clearance, the conventional measure of renal function, does not adequately reflect charges in renal drug handling in some patients, including the immunosuppressed. The aim of this study was to develop and validate a cocktail of markers. to be given in a single administration, capable of detecting alterations in the renal elimination pathways of glomerular filtration, tubular secretion and tubular reabsorption. Methods Healthy male subjects (n = 12) received intravenously infused 2500 mg sinistrin (glomerular filtration) and 440 mg p-aminohippuric acid (PAH; anion secretion), and orally administered 100 mg fluconazole (reabsorption) and 15 mg rac-pindolol (cation secretion). The potential interaction between these markers was investigated in a pharmacokinetic study where markers (M) or fluconazole (F) were administered alone or together (M + F). Validated analytical methods were used to measure plasma and urine concentrations in order to quantify the renal handling of each marker. Plasma protein binding of fluconazole was measured by ultrafiltration. All subjects had an estimated creatinine clearance within the normal range. The renal clearance of each marker (Mean +/- s.d.) was calculated as the ratio of the amount excreted in urine and thearea-under-the-concentration-time curve. Statistical comparisons were made using a paired t-test and 95% confidence intervals were reported. Results The renal clearances of sinistrin (M: 119 +/- 31 ml min(-1); M + F: 130 +/- 40 ml min(-1); P = 0.32), PAH (M: 469 +/- 145 ml min(-1); M + F: 467 +/- 146 ml min(-1); P = 0.95), R-pindolol (M: 204 +/- 41 ml min(-1); M + F: 190 +/- 41 ml min(-1); P = 0.39; n = 11), S-pindolol (M: 225 +/- 55 ml min(-1); M + F: 209 +/- 60 ml min(-1); P = 0.27; n = 11) and fluconazole (F: 14.9 +/-3.8 ml min(-1); M + F: 13.6 +/- 3.4 ml min(-1); P = 0.16) were similar when the markers or fluconazole were administered alone (M or F) or as a cocktail (M + F). Conclusions This study found no interaction between markers and fluconazole in healthy male subjects, suggesting that a single administration of this cocktail of markers of different renal processes call be used to simultaneously investigate pathways of renal drug elimination.

Nerve growth factor stimulates proliferation and survival of human breast cancer cells through two distinct signaling pathways

We show here that the neurotrophin nerve growth factor (NGF), which has been shown to be a mitogen for breast cancer cells, also stimulates cell survival through a distinct signaling pathway. Breast cancer cell lines (MCF-7, T47-D, BT-20, and MDA-MB-231) were found to express both types of NGF receptors: p140(trkA) and p75(NTR). The two other tyrosine kinase receptors for neurotrophins, TrkB and TrkC, were not expressed. The mitogenic effect of NGF on breast cancer cells required the tyrosine kinase activity of p140(trkA) as well as the mitogen-activated protein kinase (MAPK) cascade, but was independent of p75(NTR). I, contrast, the anti-apoptotic effect of NGF (studied using the ceramide analogue C2) required p75(NTR) as well as the activation of the transcription factor NF-kB, but neither p140(trkA) nor MAPK was necessary. Other neurotrophins (BDNF, NT-3, NT-4/5) also induced cell survival, although not proliferation, emphasizing the importance of p75(NTR) in NGF-mediated survival. Both the pharmacological NF-KB inhibitor SN50, and cell transfection with IkBm, resulted in a diminution of NGF anti-apoptotic effect. These data show that two distinct signaling pathways are required for NGF activity and confirm the roles played by p75(NTR) and NF-kappaB in the activation of the survival pathway in breast cancer cells.

Factors influencing job valuation: a comparative study of critical care and mon-critical care nurses

This study sought to identify the relationship between three predictor variables. perceived collaboration with medical staff, autonomy and independent actions and an outcome. the value hospital nurses placed on their work. In total 189 critical care and 366 non-critical care nurses completed a mailed survey. Critical cure nurses perceived themselves to have a mure collaborative relationship with the medical staff. described performing actions independent of medical orders more frequently and perceived their jobs to have more value than non-critical care nurses. However the latter group perceived themselves to have more autonomy in their work. Within both groups collaboration and autonomy were significantly, but weak to moderately correlated with job valuation. Simply expanding the work hospital nurses do is unlikely to result in nurses valuing their jobs more. however promoting an environment of respect and sharing between the medical and nursing staff and supporting nurses when they act in an autonomous fashion may positively influence nurses' perceptions of their work. (C) 2001 Elsevier Science Ltd. All rights reserved.

Proteomics of breast cancer for marker discovery and signal pathway profiling

Breast cancer is the most common form of cancer among women and the identification of markers to discriminate tumorigenic from normal cells, as well as the different stages of this pathology, is of critical importance. Two-dimensional electrophoresis has been used before for studying breast cancer, but the progressive completion of human genomic sequencing and the introduction of mass spectrometry, combined with advanced bioinformatics for protein identification, have considerably increased the possibilities for characterizing new markers and therapeutic targets. Breast cancer proteomics has already identified markers of potential clinical interest (such as the molecular chaperone 14-3-3 sigma) and technological innovations such as large scale and high throughput analysis are now driving the field. Methods in functional proteomics have also been developed to study the intracellular signaling pathways that underlie the development of breast cancer. As illustrated with fibroblast growth factor-2, a mitogen and motogen factor for breast cancer cells, proteomics is a powerful approach to identify signaling proteins and to decipher the complex signaling circuitry involved in tumor growth. Together with genomics, proteomics is well on the way to molecularly characterizing the different types of breast tumor, and thus defining new therapeutic targets for future treatment.

Is the irritable bladder associated with anterior compartment relaxation? A critical look at the 'integral theory of pelvic floor dysfunction'

The 'integral theory of pelvic floor dysfunction', first proposed by Petros and Ulmsten in 1990, claims that anterior vaginal wall relaxation is associated with symptoms of urgency, frequency, nocturia and urge incontinence. A retrospective study was designed to test this hypothesis. Imaging data and urodynamic reports from 272 women suffering from symptoms of lower urinary tract dysfunction were evaluated. Opening of the retrovesical angle, bladder neck descent, urethral rotation and descent of a cystocele during Valsalva were used to quantify anterior vaginal wall laxity None of the tested parameters were associated with symptoms and signs of detrusor overactivity. On the contrary, patients with higher grades of urethral and bladder descent were less likely to suffer from nocturia and urge incontinence and were less likely to leave sensory urgency and detrusor instability diagnosed on urodynamic testing. The findings of this study therefore do not support this hypothesis of the 'integral theory'.

A critical note on UNDP's gender inequality indices

This paper analyses the different indices applied for the measurement of human development as constructed by the United Nations Development Program. Of special interest is the Gender Development Index (GDI), introduced in the 1995 Human Development Report and the Gender Empowerment Measure (GEM). In light of the mate bias in the Indian socioeconomic context, the application of the GDI and GEM acquires special significance. A critical appraisal of their theoretical base and their application has been undertaken in this paper. The conclusion is that GDI and GEM. although praise-worthy achievements on the part of the UNDP, do not adequately reflect or measure male/female disparity in the Indian context. Both indices suffer from the weakness of employing a pre-assigned value of the Gender Sensitive Equity Indicator. They also exhibit several other shortcomings, outlined here. GDI is a poor indicator of the relative deprivation of females as shown by our analysis of the relationship between the GDI and the female/male ratio for 16 Indian core states.

Reduced Red Cell 2,3-Diphosphoglycerate Concentrations in Critical Illness without Decreased In Vivo P50

We investigated whether red cell 2,3-diphosphoglycerate (2,3-DPG) concentrations are reduced in critical illness, whether acidaemia, hypophosphataemia or anaemia influence 2,3-DPG, and whether there is any net effect on in vivo P50. Twenty healthy, non-smoking, male volunteers were compared with 20 male intensive care patients with APACHE 2 scores > 20 on the preceding day. Those transfused in this time were excluded. Venous red cell 2,3-DPG concentrations were measured in both groups. In the patient group, routine multichannel biochemical profile and arterial blood gas analysis were also performed and in vivo P50 calculated. The mean 2,3-DPG concentration was significantly lower in the patient group than in the controls (4.2 +/-1.3 mmoll/l vs 4.9 +/-0.5 mmol/l, P=0.016). The patients were well oxygenated (lowest arterial PO2=75 mm Hg) and showed a tendency to acidaemia (median pH 7.37, range 7.06 to 7.48) and anaemia (median haemoglobin concentration 113 g/l, range 89 to 154 g/l). By linear regression of patient data, pH had a significant effect on 2,3-DPG concentrations (r=0.6, P=0.011). Haemoglobin and phosphate concentrations did not, but there were few abnormal phosphate values. There was no correlation between 2,3-DPG concentrations and in vivo P50 (r(2) less than or equal to 0.08). We conclude that 2,3-DPG concentrations were reduced in a broad group of critically ill patients. Although this would normally reduce the P50, the reduction was primarily linked with acidaemia, which increases the P50. Overall, there was no net effect on the P50 and thus no affinity-related decrease in tissue oxygenation.

A critical appraisal of the intrinsic pancreatic angiotensin-generating system

The pancreas is a relative newcomer to the stable of tissues with an intrinsic angiotensin-generating system. The involvement of this system in pancreatic activity will be dependent on the angiotensin-generating paths present in the pancreas and their precise cellular location. Thus far, renin, angiotensin-converting enzyme (ACE), angiotensin II and AT1 and AT2 receptors have been found. These are components of the "classical" renin-angiotensin system. But there is uncertainty as to their location and site of action. Furthermore, it is not known which, if any, alternative enzymes to renin and ACE are present, which angiotensins in addition to angiotensin II are generated and whether or not there are receptors to angiotensin IV and angiotensin-(1-7). Future research should focus on these aspects in order to provide a mechanistic basis to pancreatic physiological functions and to pathological conditions of clinical relevance.

Syntaxin 7 complexes with mouse Vps10p tail interactor 1b, Syntaxin 6, vesicle-associated membrane protein (VAMP)8, and VAMP7 in B16 melanoma cells

Syntaxin 7 is a mammalian target soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) involved in membrane transport between late endosomes and lysosomes. The aim of the present study was to use immunoaffinity techniques to identify proteins that interact with Syntaxin 7. We reasoned that this would be facilitated by the use of cells producing high levels of Syntaxin 7, Screening of a large number of tissues and cell lines revealed that Syntaxin 7 is expressed at very high levels in B16 melanoma cells. Moreover, the expression of Syntaxin 7 increased in these cells as they underwent melanogenesis. From a large scale Syntaxin 7 immunoprecipitation, we have identified six polypeptides using a combination of electrospray mass spectrometry and immunoblotting. These polypeptides corresponded to Syntaxin 7, Syntaxin 6, mouse Vps10p tail interactor 1b (mVti1b), alpha -synaptosome-associated protein (SNAP), vesicle-associated membrane protein (VAMP)8, VAMP7, and the protein phosphatase 1M regulatory subunit. We also observed partial colocalization between Syntaxin 6 and Syntaxin 7, between Syntaxin 6 and mVti1b, but not between Syntaxin 6 and the early endosomal t-SNARE Syntaxin 13. Based on these and data reported previously, we propose that Syntaxin 7/mVti1b/Syntaxin 6 may form discrete SNARE complexes with either VAMP7 or VAMPS to regulate fusion events within the late endosomal pathway and that these events may play a critical role in melanogenesis.